Glaucoma is a group of heterogeneous disorders characterized by optic neuropathies, dropout of retinal ganglion cells, and visual field loss. It affects an estimated 70 million people and is the second leading cause of blindness worldwide. The exact mechanisms through which glaucoma occurs are not clearly known. Current treatments are far from satisfactory. The objective of our studies is to understand the physiopathology of glaucoma at the cellular and molecular levels and use this knowledge to develop future therapies to prevent and treat this significant blinding disease. This application explores the possibility of developing a genetic model of glaucoma in zebrafish using genetic and molecular tools. The zebrafish (Danio rerio) has become a leading model animal for the studies of human diseases because it allows powerful forward and reverse genetic analysis to be performed to quickly identify genetic loci and study their roles in the disease process. Anatomical, physiological, and genetic conservation of the important eye tissues that play key roles in maintaining intraocular pressure (IOP) in zebrafish and humans strongly suggests that the fish is a promising model animal for studying glaucoma. The annular ligament (AL) in the zebrafish eye is a specialized structure that is anatomically localized in the eye where the mammalian trabecular meshwork (TM) resides. Because TM is the primary tissue in regulation IOP, a key risk factor for glaucoma, zebrafish may develop glaucoma or glaucoma-like symptoms if its normal structure of the AL is altered by genetic ablation or modification of the AL cells. To test this hypothesis, transgenic fish will be produced by inserting three types of genes into the fish genome: a toxic gene (diphtheria toxin A, DTA), the E. coli nitroreductase (NTR) gene whose protein converts a harmless chemical compound (prodrug) into cytotoxic species, and the mutant myocilin gene that is causative for glaucoma and is known to alter the TM cells. Expression of these genes in the AL will lead to cell death or aggregation, which may result in obstruction of the aqueous humor outflow and elevated IOP. Tissue-specific cell ablation and modification is controlled by the AL-specific promoter and an inducible cre/loxP system. Histological, physiological, immunohistochemical, and molecular methods will be used to evaluate what happens to the transgenic fish in terms of overall structure of the eye, morphology in the AL, changes in IOP, cell number and viability in retinal ganglion cells, and appearance of optic nerve. The transgenic zebrafish developed in this proposal may mimic human glaucoma and provide an invaluable tool for further studying the etiology of glaucoma and for developing new drug as an affordable and high throughput system. The zebrafish (Danio rerio), a small fresh water fish and a popular model animal for the studies of human diseases, is genetically modified by selective removal or alteration of cells important to regulate normal intraocular pressure (IOP), a measurement of the fluid pressure inside the eye that helps maintain the shape of the eye. In glaucoma patients, IOP is often elevated, leading to irreversible vision loss. The fish generated in this study may have a raised IOP and develop glaucoma, providing an affordable and high throughput system for glaucoma drug and therapy development. [unreadable] [unreadable] [unreadable]